KR102251190B1 - A composition comprising extract of Syzygium formosum for skin whitening - Google Patents

Abstract

The present invention relates to a skin whitening composition containing Sijiium formosium extract as an active ingredient. The Sizigium formosium extract has excellent tyrosinase inhibitory activity and can be usefully used as a cosmetic composition for skin whitening or a composition for preventing or treating melanin hyperpigmentation disorders.

Description

Skin whitening composition comprising extract of Syzygium formosum for skin whitening {A　composition comprising extract of Syzygium formosum for skin whitening}

The present invention relates to a skin whitening composition comprising Sijiium formosium extract as an active ingredient.

The interest in skin whitening is increasingly being influenced by the increase in health and beauty orientation. As the preference for white skin is increasing due to skin damage caused by ultraviolet rays, and pursuing clearer and clearer skin with transparent makeup, interest in a method for obtaining skin whitening effect is increasing.

Skin color is influenced by various things such as melanin, blood vessel distribution and hemoglobin, the thickness of the stratum corneum and 　carotene, among which melanin plays a major role. Melanin protects the skin from ultraviolet rays and physicochemical external toxic substances, but when excessive production occurs, it not only causes serious cosmetic problems such as spots, freckles, and hyperpigmentation, but also promotes skin aging and causes skin cancer. .

Melanin is produced through the process of melanin biosynthesis in melanosomes that are inherent in melanocytes, tyrosinase, tyrosinase related protein-1 (TRP-1), and tyrosinase (TRP-2). It is regulated by melanogenic enzymes such as related 　protein-2). In more detail, α-MSH (α-melanocyte　stimulating hormone) binds to cell membrane receptors in keratinocytes and melanocytes by ultraviolet rays, increasing the amount of cyclic adenosine monophosphate (CAMP) in the cell, thereby increasing the amount of protein kinase A (PKA). ), induces the activity of tyrosinase. Tyrosinase converts tyrosine into dopa and dopaquinone, and dopaquinone passes through dopachrome in an automatic oxidative enzyme reaction 　TRP-1 and TRP-2 produce melanin. (Vincent, JH, et al., Int. J. Biochem., 19, 1141-1147, 1987).

Skin whitening is defined as brightening and whitening the skin. The mechanism of action of whitening is divided into several stages. By inhibiting or blocking the progression in the melanin synthesis stage, excessive deposition of melanin in the skin can be suppressed and the pigment content in the skin can be reduced. These methods include inhibition of tyrosinase gene expression, inhibition of glycosylation to tyrosinase enzyme, inhibition of tyrosinase enzyme activity, promotion of tyrosinase enzyme degradation, and inhibition of melanin transmission from melanocytes to keratinocytes. , Direct cytotoxicity to melanogenic cells, exfoliation and whitening by accelerating keratin formation 　 cycle. Therefore, in order to test for skin whitening, it is necessary to determine whether melanin formation is suppressed (Hearing, V. J., J Dermatol 　 Sci., 37(1), 3-14, 2005).

Ascorbic acid, kojic acid, arbutin, hydroquinone, glutathione have been previously used to treat or alleviate excessive melanin pigmentation such as spots, freckles, pigmentation, etc. due to UV exposure. (glutathione), substances with tyrosinase inhibitory activity 　 are used in cosmetics or pharmaceuticals, but they are not used due to insufficient whitening effect, safety problems on the skin, formulation and stability problems when compounded in cosmetics. Limited (Draelos, ZD, Dermatol Ther., 20(5), 308-313, 2007). Therefore, in order to solve the problems of the active ingredients, studies on the active ingredients derived from natural products with proven stability are required.

Syzygium formosum is an evergreen tree that lives in Southeast Asia such as Bangladesh, India, Myanmar, Thailand, Laos and Vietnam, and grows to a height of 10m. In Vietnam and Laos, Sijiium formosium is grown and the fruit of this tree is used for food. In addition, in Vietnam, water extract obtained by adding water to dried leaves of Sijiium formosium is used to treat atopic skin diseases and gastrointestinal disorders.

On the other hand, as a prior art related to a composition for skin whitening containing Sijiium formosium extract, the preceding paper [Thuong, PT et al., Natural Product　Sciences, 12(1), 29-37, 2006] Antioxidant activity of plant extracts including extracts has been disclosed, and Korean Patent Laid-Open No. 10-2013-0068307 discloses 15-hydroxyprostaglandin dehydrogenase (15-PGDH) containing a plant extract containing Sizigium formosium as an active ingredient. ) The composition for inhibition was disclosed, and Korean Patent No. 10-1704996 discloses a composition for the prevention or treatment of allergic diseases including Sijiium formosium extract, and the preceding paper [Adhikari, A. et al., IntJ Cosmet Sci., 30(5), 353-360, 2008] discloses the tyrosinase inhibitory activity of cloves (Syzygium aromaticum) and Java plum (Syzygium cumini).

However, there is no mention of the tyrosinase inhibitory effect, the melanin production inhibitory effect, that is, the whitening effect of the Sizigium formosium extract as in the present invention.

In addition, there is no mention of antibacterial activity and anti-atopic activity.

(Patent Document 0001) Korean Registered Patent No. 10-1704996, 　 composition for the prevention or treatment of allergic diseases containing an extract of Syzygium formosum, February 03, 2017, registered.

(Patent Document 0002) Korean Patent Laid-Open Patent No. 10-2013-0068307, Effectiveness of plant extracts

For inhibiting 15-hydroxyprostaglandin dehydrogenase (15-PGDH) contained in powder

Composition, June 26, 2013, published.

(Non-Patent Document 0001) Adhikari, A. et al., Screening of Nepalese 　 crude drugs traditionally used to treat hyperpigmentation: in vitro tyrosinase inhibition, Int J Cosmet Sci., 30(5), 353-360, 2008.

(Non-patent document 0002) Draelos, Z. D., Skin lightening preparations and the hydroquinone controversy, Dermatol Ther., 20(5), 308-313, 2007.

(Non-Patent Document 0003) Hearing, V. J., Biogenesis of pigment granules: a 　sensitive way to regulate melanocyte function, J Dermatol Sci., 37(1), 3-14, 　2005.

(Non-Patent Document 0004) Thuong, P. T. et al., Antioxidant Activities of Vietnamese Medicinal Plants, Natural Product Sciences, 12(1), 29-37, 2006.

(Non-Patent Document 0005) Vincent, J. H., et al., Mammalian tyrosinase-the 　critiacal regulatory control point in melanocyte pigmentation, Int J 　 Biochem., 19, 1141-1147, 1987.

An object of the present invention is to provide a skin whitening, antibacterial or anti-atopic composition comprising Sijiium formosium extract as an active ingredient.

In addition, it is to provide a pharmaceutical composition for preventing or improving melanin hyperpigmentation disorders comprising Sijiium formosium extract as an active ingredient.

In addition, it is to provide a pharmaceutical composition for the prevention or improvement of atopic dermatitis, comprising a Sijiium formosium extract as an active ingredient.

As a means for solving the above problems,

The present invention provides a skin whitening, antibacterial or anti-atopic cosmetic composition comprising an extract of Syzygium formosum as an active ingredient.

In addition, the Sijiium formosium extract provides a skin whitening, antibacterial or anti-atopic cosmetic composition, characterized in that sijiium formosium is extracted with 50 to 70% (v/v) of ethanol aqueous solution.

In addition, the Sijiium formosium extract is first extracted in the range of 60 ℃ to 90 ℃ with purified water, and then secondarily extracted with 50 to 70% (v / v) of ethanol aqueous solution, characterized in that skin whitening, antibacterial or anti-atopic Provides a cosmetic composition.

In addition, the Sijiium formosium extract provides a skin whitening, antibacterial or anti-atopic cosmetic composition, characterized in that the first extraction with purified water at room temperature and then second extraction with ethanol.

In addition, the Sijiium formosium extract provides a skin whitening, antibacterial or anti-atopic cosmetic composition, characterized in that it inhibits the activity of tyrosinase.

In addition, the Sijiium formosium extract provides a skin whitening, antibacterial or anti-atopic cosmetic composition, characterized in that it suppresses the production of melanin.

In addition, the Sijiium formosium extract provides a skin whitening, antibacterial or anti-atopic cosmetic composition, characterized in that it has antibacterial activity against Staphylococcus aureus.

In addition, the Sijiium formosium extract is an active ingredient, at least among madecassic acid, asian acid, corosolic acid, maslinic acid, and betulinic acid. It provides a skin whitening, antibacterial or anti-atopic cosmetic composition comprising one or more.

In addition, the active ingredient containing at least one or more of Madecassic acid, Asian acid, Corosolic acid, Maslinic acid, and Betulinic acid, It provides a skin whitening, antibacterial or anti-atopic cosmetic composition, characterized in that it is contained within the range of 10,000ppm to 200,000ppm based on the solid content of the extract formosium extract.

The present invention also provides a pharmaceutical composition for preventing or improving melanin hyperpigmentation disease comprising an extract of Syzygium formosum as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing or improving atopic dermatitis comprising an extract of Syzygium formosum as an active ingredient.

The present invention relates to a composition for skin whitening comprising Sijiium formosium extract as an active ingredient. The Sizigium formosium extract has excellent tyrosinase inhibitory activity and melanin production inhibitory activity, and thus may be usefully used as a cosmetic composition for skin whitening or a composition for preventing or improving melanin hyperpigmentation diseases.

In addition, it has excellent antibacterial and anti-atopic activity, and thus can be usefully used as an anti-bacterial and anti-atopic composition.

1 to 4 are diagrams showing the effect of improving atopic dermatitis of Sijiium formosium extract according to an embodiment of the present invention.

Hereinafter, a preferred embodiment of the present invention will be described in detail. However, the present invention is not limited to the embodiments described herein and may be embodied in other forms. Rather, the content introduced herein becomes thorough and complete, and is provided to sufficiently convey the idea of the present invention to those skilled in the art.

The skin whitening, antibacterial or anti-atopic cosmetic composition according to an embodiment of the present invention includes a Syzygium formosum extract as an active ingredient.

The Sizigium formosium extract can be obtained by extracting Sizizium formosium with water, a lower alcohol of C1 to C4 or a mixed solvent thereof, and the lower alcohol of C1 to C4 is methanol, ethanol, propanol, isopropanol, butanol and It may be selected from the group consisting of isobutanol, and is preferably an extract extracted with an aqueous ethanol solution of 50 to 70% (v/v).

In addition, the extract may be extracted with purified water in the range of 60° C. to 90° C. for the first hot water extraction, and then extracted with 50 to 70% (v/v) of ethanol solution in the range of 30° C. to 50° C.

In addition, the extract may be extracted first at room temperature with purified water and then secondly extracted at room temperature with ethanol.

As shown in the examples to be described later, the extraction content of a specific active ingredient varies according to the extraction method, and the content ratio of the specific active ingredients may vary.

The extraction time of the extract is not particularly limited, but it is preferable to extract within 10 minutes to 1 day, and as an extraction device, a conventional extraction device, an ultrasonic grinding extractor, or a fractionator may be used.

In addition, the Sizigium formosium extract exhibits a skin whitening effect by inhibiting the activity of tyrosinase.

In addition, Sijiium formosium extract was found to have excellent skin whitening effect by inhibiting melanin production.

In addition, it was confirmed that Sijiium formosium extract exhibits excellent antibacterial activity against Staphylococcus aureus. Staphylococcus aureus is known to be the cause of atopic dermatitis, and as shown in the examples to be described later, it has been confirmed that atopic dermatitis patients have an improved effect of atopic dermatitis, and have excellent anti-atopic activity.

As the main active ingredients contained in the Sizigium formosium extract, from Madecassic acid, Asian acid, Corosolic acid, Maslinic acid, and Betulinic acid. At least one or more may be included, and at least one or more of the Madecassic acid, Asian acid, Corosolic acid, Maslinic acid, and Betulinic acid is included. The active ingredient may be contained within the range of 10,000 ppm to 200,000 ppm based on the solid content of Sijiium formosium extract to provide more excellent activity.

In the cosmetic composition, Sijiium formosium extract is preferably 0.001% by weight to 50% by weight, more preferably 0.001% by weight to 40% by weight, most preferably 0.001% by weight to 30% by weight, based on the total weight of the total composition. Can be added.

The cosmetic composition may be prepared in any formulation conventionally prepared in the art, for example, solutions, emulsions, suspensions, pastes, creams, lotions, gels, powders, sprays, surfactant-containing cleansing, Oil, soap, liquid cleaner, bathing agent, foundation, makeup base, essence, lotion, foam, pack, softening water, sunscreen 　 cream or sun oil can be prepared in a formulation selected from the group consisting of, but is not limited thereto.

In addition, the cosmetic composition may include all of the ingredients commonly used in cosmetics, in addition to the Sijiium formosium extract. For example, it may contain general auxiliary ingredients such as emulsifiers, thickeners, emulsions, surfactants, lubricants, alcohols, water-soluble polymers, gelling agents, stabilizers, vitamins, inorganic salts, emulsifiers, and fragrances. According to the formulation or purpose of use, the 　 ingredients can be added within a range that does not impair the inherent effect of the cosmetic.

The cosmetically acceptable 　 carrier included in the cosmetic composition of the present invention varies depending on the formulation of the cosmetic composition.

When the formulation of the present invention is a solution or emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component. For example, water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol , 1,3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan, and the like may be used.

When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, an ethoxylated isostearyl alcohol, a suspending agent such as polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, micro Crystalline cellulose, aluminum metahydroxide, bentonite, or tracant may be used.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, etc. are used as carrier components. Can be.

When the formulation of the present invention is a powder or spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, etc. may be used as a carrier component. Propellants such as locarbon, propane/butane or dimethyl ether.

When the formulation of the present invention is a surfactant containing cleansing, as a carrier component, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol 　 fatty acid ester, and the like may be used.

The cosmetic composition containing the Sizigium formosium extract can be used daily and can be used for an indefinite period of time, preferably 　the amount of use depending on the age, skin condition or skin type of the user, and the concentration of the Sizigium formosium extract 　, You can adjust the frequency and period of use.

In yet another aspect, the present invention relates to a pharmaceutical composition for preventing or improving melanin hyperpigmentation disease, comprising a Sijiium formosium extract as an active ingredient. In addition, the present invention relates to a pharmaceutical composition for preventing or improving atopic dermatitis comprising an extract of Syzygium formosum as an active ingredient.

The melanin hyperpigmentation disease is a group consisting of freckles, liver spots, melasma, brown or dark spots, gestational brown spots, senile spots, spots caused by UV exposure, hyperpigmentation after inflammation due to wounds or dermatitis, and hyperpigmentation after drug use. It may be a disease selected from.

Atopic dermatitis is a chronic recurring skin disease, often accompanied by itching, and the etiology of atopic dermatitis is not yet clear, but it is caused by an immunological abnormality mediated by type 2 T auxiliary lymphocytes. It is known to be caused by a genetic predisposition, psychological factor, or pharmacological factor, but is included in atopic dermatitis defined in the present invention, but is not limited to the above description.

The pharmaceutical composition according to the present invention may be formulated in a suitable form together with a generally used pharmaceutically acceptable carrier. "Pharmacologically acceptable" refers to a composition that is physiologically acceptable and, when administered to a human, does not usually cause allergic reactions or similar reactions such as gastrointestinal disorders, dizziness and the like.

In addition, the pharmaceutical compositions may be formulated and used in the form of oral dosage forms such as powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols, etc., skin external preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. have. Carriers, excipients and diluents that may be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, gum arabic, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, Methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methyl paraoxybenzoate, propyl paraoxybenzoate, talc, magnesium stearate, and mineral oil may be included, but are not limited thereto. When formulated, it is prepared using diluents or excipients such as commonly used fillers, stabilizers, binders, disintegrants, and surfactants. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and these solid preparations include at least one excipient in the extract of the present invention, such as starch, calcium carbonate, sucrose, or lactose, gelatin. It is prepared by mixing the back. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use include suspensions, liquid solutions, emulsions, syrups, etc. In addition to water and liquid paraffin, which are commonly used simple diluents, various excipients such as humectants, sweeteners, fragrances, preservatives, etc. may be included. . Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried formulations, and suppositories. As the non-aqueous solvent and suspending agent, propylene 　 glycol, polyethylene glycol, vegetable oil such as olive oil, and injectable ester such as ethyl oleate may be used. As a base for suppositories, witepsol, macrogol, tween 61, cacao butter, laurin paper, glycerogelatin, and the like can be used.

The pharmaceutical composition comprising the Sizigium formosium extract disclosed in the present invention as an active ingredient may be administered to mammals such as mice, livestock, and humans by various routes. All modes of administration can be expected and may be administered, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or cerebrovascular injection. The dosage is the age, sex, weight of the subject to be treated, the specific disease or pathology to be treated, the severity of the disease or pathology, the time of administration, the route of administration, the rate of absorption, distribution and excretion of the drug, the type of other drugs used, and the prescriber's It will depend on your judgment, etc. Dosage determination based on these factors is within the level of those skilled in the art, and dosages generally range from 0.001 mg/kg/day to 2000 mg/kg/day. A more preferred dosage is from 0.01 mg/kg/day to 500 mg/kg/day. Administration may be administered once a day, or may be divided several times. The above dosage does not limit the scope of the present invention in any way.

<Example 1 and Example 2. Preparation of Sijiium formosium extract>

A 50% (v/v) 　 ethanol aqueous solution or 200 ml of a 70% (v/v) ethanol aqueous solution was added to 20 g of Syzygium formosum dry powder, followed by extraction at 40° C. for 2 hours. The extract obtained from the above process was filtered and concentrated to obtain a Siziziium formosium 50% ethanol aqueous solution extract of Example 1 and a Siziziium formosium 70% ethanol aqueous solution extract of Example 2.

<Comparative Example 1. Preparation of Sijiium formosium 10% ethanol aqueous solution extract>

Prepared as in Example 1, but using a 10% ethanol 　 aqueous solution instead of 50% ethanol aqueous solution to obtain a Sijiium formosium 10% ethanol aqueous solution extract.

<Comparative Example 2. Preparation of Sijiium Formosium 20% Ethanol Aqueous Solution Extract>

Prepared as in Example 1, but using a 20% ethanol 　 aqueous solution instead of 50% ethanol aqueous solution to obtain a Sijiium formosium 20% ethanol aqueous solution extract.

<Comparative Example 3. Preparation of Sijiium Formosium 40% Ethanol Aqueous Solution Extract>

Prepared as in Example 1, but using a 40% ethanol 　 aqueous solution instead of 50% ethanol aqueous solution to obtain a Sijiium formosium 40% ethanol aqueous solution extract.

<Comparative Example 4. Preparation of Sijiium Formosium 80% Ethanol Aqueous Solution Extract>

Prepared as in Example 1, but using 80% ethanol 　 aqueous solution instead of 50% ethanol aqueous solution to obtain a Sijiium formosium 80% ethanol aqueous solution extract.

<Comparative Example 5. Preparation of Sijiium Formosium Ethanol Extract>

Prepared as in Example 1, but using 100% ethanol instead of 50% ethanol aqueous solution to obtain a Sijiium formosium 100% ethanol extract.

<Comparative Example 6. Preparation of Sijiium Formosium Methanol Extract>

Prepared as in Example 1, but using 100% methanol instead of 50% ethanol aqueous solution to obtain a 100% methanol extract of Sijiium formosium.

<Comparative Example 7. Preparation of Sijiium Formosium Hot Water Extract>

Prepared as in Example 1, but extracted for 1 hour at 80 °C using water instead of a 50% aqueous ethanol solution to obtain a Sizigium formosium hot water extract.

<Experimental Example 1. Confirmation of tyrosinase inhibitory activity using L-tyrosine>

The inhibitory activity of tyrosinase, an enzyme that plays an important role in the melanin synthesis process, was confirmed using the present invention Sijiium formosium extract.

500 μl of 1 mM L-tyrosine, 900 μl of 0.1 M phosphate buffer (pH 6.8) and 0.5 mg/ml Sizigium formosium extract dissolved in 10% DMSO (Example 1, Example 2, Comparative Example 1 to 7) 100 μl of each were mixed, and then 100 μl of tyrosinase (53.7 U/ml) was added and reacted at 25° C. for 15 minutes. At this time, 　DMSO was used as a control group, and 0.5mg/ml arbutin was used as a positive control group.

The reaction solution was measured for absorbance at 475 nm for 15 minutes at 30 second intervals, and then the tyrosinase inhibitory activity (%) was calculated by Equation 1 below, and is shown in Table 1.

[Equation 1]

Condition Inhibitory activity (%) Example 1 50% ethanol aqueous solution extract 75.0 Example 2 70% ethanol aqueous solution extract 70.8 Comparative Example 1 10% ethanol aqueous solution extract 25.3 Comparative Example 2 20% ethanol aqueous solution extract 31.2 Comparative Example 3 40% ethanol aqueous solution extract 40.0 Comparative Example 4 80% ethanol aqueous solution extract 54.5 Comparative Example 5 100% ethanol extract 40.7 Comparative Example 6 100% methanol extract 30.4 Comparative Example 7 Hot water extract 20.0 Positive control Arbutin 49.1 Control DMSO 0

Looking at Table 1, it was confirmed that the Sijiium formosium 　 50% and 70% ethanol aqueous solution extracts of Examples 1 and 2 of the present invention had superior tyrosinase inhibitory activity compared to arbutin, a positive control. In particular, the examples of the present invention showed remarkably excellent tyrosinase inhibitory activity compared to the Sizigium formosium extracts of Comparative Examples 1 to 7 under different solvent conditions.

In addition, although not shown in Table 1, the tyrosinase inhibitory activity of Clove (Syzygium aromaticum) and Java Plum (Syzygium cumini) was typically confirmed among the homogeneous heterogeneous species of Sizigium formosium used in the present invention. And it was found that the tyrosinase inhibitory activity was 20 times or more 　 lower than that of Sijiium formosium extract of 2, and it was found that the difference in the tyrosinase inhibitory activity value was different even in plants of the same genus.

Therefore, from the above results, it was found that the Sizigium formosium extract of Examples 1 and 2 of the present invention can be usefully used as a composition exhibiting skin whitening effect by inhibiting the formation of melanin in the skin.

<Experimental Example 2. Confirmation of tyrosinase inhibitory activity using L-DOPA>

Using L-DOPA, tyrosinase inhibitory activity was confirmed.

(1) Preparation of reagents

-Tyrosinase solution: Tyrosinase was dissolved in 0.1M PBS (Phosphate buffered saline) buffer to prepare a solution at a concentration of 110 U/ml.

-Substrate: L-DOPA was dissolved in 0.1M PBS buffer to make 5mM.

-Use of sample: It was used after diluting to the corresponding concentration with distilled water.

-Negative control: Distilled water was added instead of the sample.

(2) Test method

Sijiium formosium extract (Example 1, Example 2, Comparative Examples 1 to 7) and negative control (control), positive control (positive control) 30 µl, 0.1M PBS buffer 70 µl, and substrate 30 µl were added. After that, 20 µl of tyrosinase is added and reacted at 25°C for 5 minutes. After completion of the reaction, absorbance was measured at 475 nm. By comparing the absorbance of the test group compared to the control group, the tyrosinase inhibitory activity (%) was calculated by Equation 1, and is shown in Table 2.

Condition Inhibitory activity (%) Example 1 50% ethanol aqueous solution extract 32.3 Example 2 70% ethanol aqueous solution extract 46.2 Comparative Example 1 10% ethanol aqueous solution extract 8.7 Comparative Example 2 20% ethanol aqueous solution extract 15.9 Comparative Example 3 40% ethanol aqueous solution extract 24.8 Comparative Example 4 80% ethanol aqueous solution extract 28.5 Comparative Example 5 100% ethanol extract 27.7 Comparative Example 6 100% methanol extract 24.4 Comparative Example 7 Hot water extract 8.5 Positive control Arbutin 18.6 Control Distilled water 0

Looking at Table 2, it was confirmed that the Siziziium formosium 　 50% and 70% ethanol aqueous solution extracts of Examples 1 and 2 of the present invention had superior tyrosinase inhibitory activity compared to arbutin, a positive control.

<Experimental Example 3. Confirmation of melanin production inhibitory activity>

(1) cell culture

The cell line used in the test is the B16F10 cell line (Korea Cell Line Bank, Korea), which is a mouse malignant melanoma cell. The cryopreserved cell line was cultured in a culture dish, and after the first cell culture, passages were repeated 2 to 3 times every 2 to 3 days to confirm the stabilization of the cell line, and then cultured in a 48 well plate as many as 1×105/well cells. . After that, the test was prepared by incubating for 24 hours.

(2) Sample preparation

Prepare a test by dissolving Sijiium formosium extract (Example 1, Example 2, Comparative Examples 1 to 7) at a concentration of 0.1 mg/ml in DMEM (Dimethyl modified eagle medium, 10-013-CVR, Corning, USA) I did.

(3) Test method

In a 48 well plate in which the cells were cultured in advance, 300 µl of the sample and the medium containing the negative control group and the positive control group were added each, and cultured with medium exchange every day under conditions of 37°C and 5% CO2 for 72 hours. Thereafter, each well was washed with PBS to remove the medium, and then 200 µl of 1N NaOH was treated. After that, the absorbance was measured at 400 nm using SpectraMax M2 (Molecular devices, USA). The degree of inhibition of melanin production was calculated as shown in Equation 2 below by comparing the absorbance of the test group compared to the control group treated with the negative control group, and the results are shown in Table 2 below.

[Equation 2]

Condition Inhibitory activity (%) Example 1 50% ethanol aqueous solution extract 22.8 Example 2 70% ethanol aqueous solution extract 28.6 Comparative Example 1 10% ethanol aqueous solution extract 5.6 Comparative Example 2 20% ethanol aqueous solution extract 10.5 Comparative Example 3 40% ethanol aqueous solution extract 18.6 Comparative Example 4 80% ethanol aqueous solution extract 20.6 Comparative Example 5 100% ethanol extract 19.2 Comparative Example 6 100% methanol extract 18.9 Comparative Example 7 Hot water extract 4.7 Positive control Arbutin 21.1 Control DMEM 0

Therefore, from the above results, it was found that the Sizigium formosium extract of Examples 1 and 2 of the present invention can be usefully used as a composition exhibiting skin whitening effect by inhibiting the formation of melanin in the skin.

<Experimental Example 4. Antibacterial activity confirmation>

Antimicrobial activity was tested using the Sizigium formosium extract of Example 2. As the target strain, Staphylococcus aureus (Staphylococcus aureus , hereinafter S. aureus ), also known as a skin opportunistic infectious bacteria and also known as atopic causative bacteria, was selected to confirm antimicrobial and anti-atopic activity.

(1) Preparation of S. aureus strain

First, after culturing the staphylococcus aureus (Staphylococcus aureus, or less S. aureus) strains in 37 ℃, 200rpm in LB media using a physiological saline solution was diluted with bacterial count of 8x10 ^ 6 cfu / ml were prepared for strain.

(2) Preparation of Sijiium formosium extract

Sizigium formosium extract of Example 2 was dissolved in DMSO, diluted, and 30µl was added to LB and finally 0.1, 0.2, 0.25, 0.4, 0.5, 0.6, 0.75, 1, 1.5 mg/ml An extract sample was prepared.

(3) Antibacterial activity test

After inoculating 30 µl of S. aureus strain in the Sizigium formosium extract sample to make the number of bacteria 8x10^6 cfu/ml, cultured for 12 hours at 37° C. and 120 rpm, and measuring the absorbance at 595 nm for antimicrobial activity. Was tested. 5% DMSO was used as a control, and the results are shown in Table 4 below.

division control
(final
5% DMSO) Sisium formosium (mg/ml) 0.1 0.25 0.5 0.75 One 1.5 One 0.656 0.596 0.489 0.439 0.265 0.208 -0.065 2 0.696 0.606 0.426 0.268 0.421 0.304 0.272 3 0.821 0.627 0.555 0.345 0.453 0.148 -0.203 4 0.657 0.593 0.499 0.435 0.312 0.190 0.331 5 0.723 0.648 0.529 0.378 0.295 0.237 0.029 average 0.711
±
0.068 0.614
±
0.023 0.500
±
0.048 0.373
±
0.071 0.349
±
0.083 0.217
±
0.058 0.073
±
0.226

As shown in Table 4, it can be seen that the Sizigium formosium extract according to an embodiment of the present invention has excellent antibacterial activity and anti-atopic activity.

<Examples 3 to 6. Confirmation of components of Sijiium formosium extract according to extraction conditions>

<Example 3>

200 ml of an aqueous solution of 70% (v/v) 　 ethanol was added to 20 g of dry powder of Syzygium formosum, followed by extraction at room temperature for 2 hours. The extract obtained from the above process was filtered and concentrated to obtain a Sijiium formosium extract.

<Example 4-1>

200 ml of purified water was added to 20 g of dry powder of Syzygium formosum, extracted at 80° C. for 1 hour, filtered, and concentrated to obtain a Sizygium formosium extract.

<Example 4-2>

200 ml of an aqueous solution of 50% (v/v) 　 ethanol was added to Sijiium formosium filtered in Example 4-1, followed by extraction at 40° C. for 2 hours. The extract obtained from the above process was filtered and concentrated to obtain a Sijiium formosium extract.

<Example 4-3>

200 ml of an aqueous solution of 70% (v/v) 　 ethanol was added to Sijiium formosium filtered in Example 4-2, followed by extraction at 40° C. for 2 hours. The extract obtained from the above process was filtered and concentrated to obtain a Sijiium formosium extract.

<Example 5-1>

200 ml of an aqueous solution of 70% (v/v) 　 ethanol was added to 20 g of dry powder of Syzygium formosum, followed by extraction at room temperature for 1 hour. The extract obtained from the above process was filtered and concentrated to obtain a Sijiium formosium extract.

<Example 5-2>

200 ml of an aqueous solution of 70% (v/v) 　 ethanol was added to the Sijiium formosium filtered in Example 5-1, followed by extraction at room temperature for 1 hour. The extract obtained from the above process was filtered and concentrated to obtain a Sijiium formosium extract.

<Example 5-3>

200 ml of an aqueous solution of 70% (v/v) 　 ethanol was added to the Sijiium formosium filtered in Example 5-2, followed by extraction at room temperature for 1 hour. The extract obtained from the above process was filtered and concentrated to obtain a Sijiium formosium extract.

<Example 6-1>

200 ml of purified water was added to 20 g of dry powder of Syzygium formosum, extracted for 30 minutes at room temperature, filtered, and concentrated to obtain a Sizygium formosium extract.

<Example 6-2>

About 200 ml of 100% ethanol was added to Sijiium formosium filtered in Example 4-1, followed by extraction at room temperature for 2 hours. The extract obtained from the above process was filtered and concentrated to obtain a Sijiium formosium extract.

<Experimental Example 5. Confirmation of components of Sijiium formosium extract using HRMS analysis>

(1) HRMS analysis conditions

HRMS analysis was performed under the conditions of Tables 5 and 6 below.

HRMS condition Column YMC-Triart C18
(5 um 250 X 4.6 mm) Solvent (A) 0.1% Formic acid in water
(B) 0.1% Formic acid in CAN Flow rate 0.5 ml/min Injection volume 10 ul

Time(min) A(%) B(%) 0 75 25 25 60 40 65 10 90 66 0 100 70 0 100 75 75 25 80 75 25

** solvent gradient over time

(2) Quantification result of major active ingredients

The main active ingredients of the Sizigium formosium extract of Example 3 were identified, and the weight content measurement results are shown in Table 7 below based on the solid content of the extract.

Main active ingredient Content (PPM) Madecassic acid 16000 Asiatic acid 15000 Corosolic acid 48000 Maslinic acid 36000 Betulinic acid 12000

(3) Analysis result of effective ingredient extraction efficiency according to extraction conditions

The efficiency of extracting active ingredients according to the extraction conditions of Examples 3 to 6 was analyzed by the HRMS method.

division Madecassic acid Asiatic acid Corosolic acid Maslinic acid Betulinic acid Example 3 100 100 100 100 100 Example 4-1 2 20 0 0 0 Example 4-2 16 71 55 53 23 Example 4-3 14 78 31 29 22 Example 5-1 21 60 40 38 35 Example 5-2 32 0 0 0 31 Example 5-3 26 0 0 0 11 Example 6-1 2 15 0 0 0 Example 6-2 20 52 30 28 35

<Formulation Example 1. Preparation of cosmetic composition>

Formulation Example 1-1. Preparation of flexible lotion

Using the Sijiium formosium 50% ethanol aqueous solution extract of Example 1 of the present invention, a flexible lotion was prepared according to the composition shown in Table 9 below.

Raw material name Content (% by weight) Example 1 4.0 Butylene glycol 3.5 glycerin 2.5 Polyoxyethylene hydrogenated castor oil 0.1 ethanol 2.5 Betaine 1.0 Citric acid 0.01 Sodium citrate 0.03 antiseptic Appropriate amount Spices Appropriate amount Purified water to 100

Formulation Example 1-2. Preparation of nourishing essence

Using the Sijiium formosium 50% ethanol aqueous solution extract of Example 1 of the present invention, a nutritional essence was prepared by a conventional method according to the composition shown in Table 10 below.

Raw material name Content (% by weight) Example 1 7.0 Cetostearyl alcohol 1.0 Self-emulsifying monostearic acid 1.0 Beeswax 0.5 Squalane 5.0 Isocetyloctanoate 3.0 Dimethylsiloxane 0.3 Sorbitan monostearate 0.5 Polyethylene glycol monostearate 8.0 glycerin 4.0 Propylene glycol 0.2 Carboxypolymer 0.22 Triethanolamine 0.25 antiseptic Appropriate amount Spices Appropriate amount flash Appropriate amount Purified water to 100

Formulation Example 1-3. Preparation of cream

Using the Sijiium formosium 50% ethanol aqueous solution extract of Example 1 of the present invention, a cream was prepared in a conventional manner according to the composition shown in Table 11 below.

Raw material name Content (% by weight) Example 1 7.0 Cetostearyl alcohol 3.0 Self-emulsifying monostearic acid 1.5 Lipophilic monostearic acid 1.5 Beeswax 0.5 Floating paraffin 8.0 Squalane 7.0 Isocetyloctanoate 4.0 Refined Jojoba Oil 4.0 Dimethylsiloxane 0.3 Sorbitan monostearate 1.0 Polyethylene glycol monostearate 1.2 glycerin 6.0 Propylene glycol 4.0 Betaine 4.0 Xanthan gum 0.06 Triethanolamine 0.10 antiseptic 0.25 Spices Appropriate amount flash Appropriate amount Purified water to 100

<Experimental Example 6. Anti-Atopy Test>

Targeting patients with atopic dermatitis acutely worsened, the cream of Formulation Example 1-3 was applied to the affected area of atopic 　 patients three times a day, and then the effect of improving atopic dermatitis was confirmed, and the results are shown in Figs. It is shown in 4. As can be seen, it can be confirmed that the atopic symptoms have improved significantly.

Claims (11)

Contains Syzygium formosum extract as an active ingredient,
As the active ingredient, a skin whitening cosmetic comprising at least one or more of Madecassic acid, Asian acid, Corosolic acid, Maslinic acid, and Betulinic acid Composition. The method of claim 1,
The Sijiium formosium extract is a skin whitening cosmetic composition, characterized in that the Sijiium formosium is extracted with 50 to 70% (v/v) ethanol aqueous solution. The method of claim 1,
The Sijiium formosium extract is a skin whitening cosmetic composition, characterized in that after the first extraction in the range of 60 ℃ to 90 ℃ with purified water, and the second extraction with 50 to 70% (v / v) of ethanol aqueous solution. The method of claim 1,
The Sijiium formosium extract is a skin whitening cosmetic composition, characterized in that the first extraction at room temperature with purified water, and then second extraction with ethanol. The method of claim 1,
The Sijiium formosium extract is a skin whitening cosmetic composition, characterized in that it inhibits the activity of tyrosinase (tyrosinase). The method of claim 1,
The Sijiium formosium extract is a skin whitening cosmetic composition, characterized in that it suppresses the production of melanin. delete delete The method of claim 1,
The active ingredient containing at least one or more of the madecassic acid, asian acid, corosolic acid, maslinic acid, and betulinic acid Skin whitening cosmetic composition, characterized in that contained within the range of 10,000ppm to 200,000ppm based on the solid content of the mosium extract. A pharmaceutical composition for preventing or improving melanin hyperpigmentation disorders comprising an extract of Syzygium formosum as an active ingredient. delete

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